Our research is aimed at genetic elucidation of the control of differentiation in cultured mouse melanoma cells. Results of the past year that underscore the feasibility of applying gene transfer methods in the melanoma system have led us to focus increasingly on approaches made possible by this powerful technology. Three clonal cell populations diverging widely in their expression of melanocyte traits are all competent as recipients for transfer of plasmid-cloned selectable genes by calcium phosphate-coprecipitated DNA. Pigmented dendritic clone B559 cells and unpigmented fibroblast-like cells from bromodeoxyuridine-treated B559 (clone C3471) approach LTK- in plasmid-dependent transferent colony frequencies. Spontaneously amelanotic epithelioid clone B78H1 exhibits competence superior to that of LTK-: 100 colonies/ng of plasmid, and a maximum frequency of 10-2/plated recipient. Our efforts to employ gene transfer as a bioassay for sequences determining screenable differentiated traits have led to success in transferring a gene from total human melanoma DNA that causes B78H1 recipients to display a human melanoma antigen. In collaboration with T. Albino and L. Old we are performing further experiments preparatory to cloning this human gene. We are also continuing attempts to transfer genes encoding pigment or plasminogen activator into the nonexpressing B78H1 cells. We have used cotransformation to introduce cloned genomic genes for peptide hormones into B559, C3471, and B78H1 and are now analyzing expression of these genes in these disparate backgrounds. We are also following up the observation that 5-azacytidine (AzaCr) treatment causes B78H1 cells to express melanocytic phenotypes, by analyzing cell DNA methylation patterns in AsaCr-treated and untreated melanoma cells.